We have created a nonmuscle myosin heavy chain II-A (NMHC II-A) null mouse in order to study the function of this myosin II isoform and its relation to the other nonmuscle myosin isoforms in its class (NMHC II-B and II?C). Exon 3, near the ATP binding region of the myosin heavy chain, was targeted by insertion of the neomycin cassette. In contrast to the NMHC II-B null mouse which dies around E12 to birth with severe defects in the heart and brain, the deletion of NMHC II-A results in lethality at E7.5 or earlier. NMHC II-A null embryos are smaller than wild-type or heterozygous littermates and are severely disorganized. Embryonic stem (ES) cells express NMHC II-A and II-B, but not II-C. A-/A- ES cells were generated by re-electroporation of A+/A- cells followed by selection at increased concentrations of G418. Because of the early lethality of the NMHC II-A null mice, embryoid bodies which recapitulate the early stages of embryonic development were formed from these cells and studied along with A+/A- and wild-type embryoid bodies. Conventional RT-PCR and real time RT-PCR showed that some markers of visceral endoderm specification are present in all three types of embryoid bodies. However, a number of markers of visceral endoderm and mesoderm development are missing or reduced in the null embryoid bodies. We are culturing blastocysts (E3.5) from A+/A- intercrosses in an effort to determine the earliest stage at which the embryos begin to die. We are also making chimeric embryos by injecting A+/A- and A-/A- ES cells into Rosa 26 blastocysts in an effort to rescue the defect in the null embryos. Attempts to rescue A-/A- ES cells by introduction of GFP tagged NMHC II-A have, to date, been unsuccessful. New constructs have been made to introduce the neomycin cassette into an intron, creating a hypomorph which expresses low levels of NMHC II-A in order to study the function of myosin II-A. And finally, we have created a NMHC II-A bearing a mutation which mimics one found in certain human disease states.